Processes for preparing β-lactams

ABSTRACT

The present invention relates to hitherto unknown compounds of the general formula I: ##STR1## in which R 1  stands for a phenyl, 4-hydroxyphenyl, 1,4-cyclohexadienyl or a 3-thienyl group; R 2  represents a primary amino or a carboxy group; R 3  is a hydrogen atom, or a lower alkyl, aryl or aralkyl radical, and A stands for a radical of a β-lactamase inhibitor containing a β-lactam ring as well as a carboxy group, A being connected via the carboxy group. 
     The present invention provides new compounds useful in the treatment of bacterial infections. The new compounds are in particular strongly active against β-lactamase producing bacteria.

This is a continuation of application Ser. No. 213,083, filed Dec. 4,1980, now U.S. Pat. No. 4,342,772, which is itself acontinuation-in-part of Ser. No. 118,063, filed Jan. 24, 1980.

The present invention relates to hitherto unknown β-lactamase inhibitorcontaining a β-lactam ring as well as a carboxy group; A being connectedvia the carboxy group. More specifically, A is represented by one of thegeneral formulae II, III, or IV: ##STR2## in which R₄ stands for ahydrogen or a halogen atom; R₅ is a hydrogen atom or an amino oracylamino group, but at least one of R₄ and R₅ being hydrogen; R₆represents a halogen atom; and R₇ stands for a hydroxyl group, or one ofthe radicals of known clavulanic acid derivatives with β-lactamaseinhibitory activity.

Generally, "lower alkyl" stands for a C-1 to C-6 straight or branchedalkyl radical, aryl stands for a monocyclic or bicyclic, carbocyclicradical, and acylamino stands for a radical present in the side chain ofwell-known penicillins. The asterisk in the side chain and, in case R₃is different from hydrogen, the dagger in the ester moiety indicatechiral centers which give rise to diastereomeric forms of the compoundsof formula I. The invention comprises all such diastereomers as well asmixtures thereof.

The salts of the new compounds are salts with pharmaceuticallyacceptable, non-toxic acids or bases, depending on whether R₂ stands fora primary amino group or for a carboxy group.

Among suitable acids can be mentioned hydrochloric acid, hydrobromicacid, hydroiodic acid, phosphoric acid, sulphuric acid, nitric acid,p-toluenesulphonic acid, methanesulphonic acid, formic acid, aceticacid, propionic acid, citric acid, tartaric acid, maleic acid, pamoicacid, and p-(dipropylsulfamyl)benzoic acid (probenecid). Among suitablebasic salts can be mentioned alkali metal salts or alkaline earth metalsalts, such as sodium, potassium, magnesium, or calcium salts as well assalts with ammonia or suitable non-toxic amines, such as loweralkylamine, e.g. triethylamine, hydroxy-lower alkylamines, for example2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine ortris-(2-hydroxyethyl)-amine, cycloalkylamines, for exampledicyclohexylamine, or benzylamines, for exampleN,N'-dibenzylethylenediamine or dibenzylamine, without these examplesbeing limiting the invention. Also salts with acidic or basicantibiotics are within the scope of the invention. In some instances, itis preferred to use easily soluble salts, whereas for other purposes, itmay be appropriate to use an only slightly soluble salt, e.g. in orderto obtain a prolonged effect. In particular, a prolonged effect can beobtained by using a salt with probenecid which blocks the tubularexcretion of β-lactam compounds.

In the clinical treatment of bacterial infections it is a seriousproblem that β-lactamase producing bacteria are occurring withincreasing frequency. These enzymes inactivate most penicillins andcephalosporins, and it is well recognized that β-lactamases from bothgram-positive and gram-negative bacteria contribute significantly to theresistance of bacteria to β-lactam antibiotics.

Several naturally occurring β-lactamase inhibitors, includng clavulanicacid and the olivanic acids, have been described. More recently, anumber of semisynthetic β-lactam compounds, e.g. penicillanic acid1,1-dioxide, 6α-chloropenicillanic acid 1,1-dioxide, a series ofclavulanic acid derivatives, 6β-bromopenicillanic acid, methicillinsulphone, and quinacillin sulphone, were found to possess similarbiological properties. With a few exceptions, these compounds displayonly weak antibacterial activity against most gram-positive andgram-negative organisms, but are powerful inhibitors of a wide range ofβ-lactamases. In combination with selected penicillins andcephalosporins, the compounds act synergistically against a variety ofβ-lactamase producing bacteria because they protect the penicillins andcephalosporins against inactivation.

As mentioned above, the present invention provides new compounds inparticular intended for enteral use and being strongly antibacteriallyactive in vivo. The advantageous effect against β-lactamase producingbacteria is achieved because the compounds contain in one and the samemolecule both the moiety of an antibacterially highly active penicillinand the moiety of a potent β-lactamase inhibitor. However, twoprerequisites are necessary to utilize this feature of the newcompounds. They must be capable of being absorbed from thegastro-intestinal tract, and during or after the absorption they must behydrolyzed with liberation of the penicillin and the β-lactamaseinhibitor. It has turned out that both of these prerequisites arefulfilled, and therefore the present compounds are valuable pro-drugs ofboth the penicillins and the β-lactamase inhibitors.

Thus, studies in animals and human volunteers have shown that the newcompounds are readily absorbed from the gastro-intestinal tract. Duringor after the absorption they are hydrolyzed with liberation of equimolaramounts of the two components in question, the penicillin and theβ-lactamase inhibitor, giving rise to simultaneous high blood and tissuelevels of the two components. Thereby the penicillins are protectedagainst inactivation by the β-lactamases.

The efficient absorption and in vivo hydrolysis of the compounds of theinvention are illustrated by a study in human volunteers dosed orallywith one of the new compounds, namely the hydrochloride of1,1-dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate, in the following calledVD-1827. For comparison, the same group of volunteers was also givenequimolar amounts of the orally active ampicillin pro-drug,pivampicillin, and potassium penicillanate 1,1-dioxide, respectively.The results of these studies are summarized in Tables I and II.

                                      TABLE I                                     __________________________________________________________________________    Serum concentrations and urinary excretion of ampicillin in fasting           volunteers                                                                    following oral administration of                                              A. 125 mg of pivampicillin free base in tablets.                              B. 170 mg of VD-1827.sup.(x) hydrochloride (corresponding to 125 mg of        pivampicillin                                                                 free base) in aqueous solution.                                                                                   Urinary excretion                         Serum concentrations (μg/ml)    (% of dose)                                Hours after administration                                                    0.25       0.5   1     2     4     0-6   0-24                                 Subject                                                                           A   B  A  B  A  B  A  B  A  B  A   B A   B                                __________________________________________________________________________    GK  0.22                                                                              1.6                                                                              2.2                                                                              2.1                                                                              3.7                                                                              1.8                                                                              1.2                                                                              0.48                                                                             0.13                                                                             0.09                                                                             78  57                                                                              79  58                               MK  <0.03                                                                             2.7                                                                              1.1                                                                              3.3                                                                              3.7                                                                              1.7                                                                              1.5                                                                              0.52                                                                             0.17                                                                             0.06                                                                             76  60                                                                              76  61                               FJ  0.13                                                                              1.2                                                                              2.3                                                                              3.3                                                                              2.3                                                                              2.4                                                                              1.1                                                                              0.52                                                                             0.17                                                                             0.09                                                                             50  65                                                                              52  66                               MM  0.34                                                                              1.9                                                                              3.1                                                                              2.9                                                                              3.7                                                                              2.3                                                                              1.7                                                                              0.63                                                                             0.27                                                                             0.13                                                                             NS.sup.(+)                                                                        54                                                                              NS.sup.(+)                                                                        57                               LA  1.7 1.4                                                                              3.1                                                                              4.0                                                                              3.4                                                                              2.6                                                                              1.2                                                                              1.0                                                                              0.23                                                                             0.25                                                                             64  66                                                                              66  70                               Mean                                                                              (0.48)                                                                             1.76                                                                             2.36                                                                             3.12                                                                             3.36                                                                             2.16                                                                             1.34                                                                            0.63                                                                             0.19                                                                             0.12                                                                             67  60                                                                              68  62                               __________________________________________________________________________     .sup.(x) VD1827 is 1,1dioxopenicillanoyloxymethyl                             6(D-amino-phenylacetamido)penicillanate                                       .sup.(+) No sample                                                       

                  TABLE II                                                        ______________________________________                                        Urinary excretion in 0 to 6 hours of penicillanic acid                        1,1-dioxide in fasting volunteers following oral adminis-                     tration of                                                                    A. 73 mg of potassium penicillanate 1,1-dioxide                               (corresponding to 63 mg of penicillanic acid                                  1,1-dioxide) in aqueous solution                                              B. 170 mg of VD-1827 hydrochloride (corresponding                             to 63 mg of penicillanic acid 1,1-dioxide) in                                 aqueous solution                                                                              Urinary excretion (% of dose)                                 Subject           A     B                                                     ______________________________________                                        GK                2.5   60                                                    MK                4.0   76                                                    FJ                9.5   77                                                    MM                5.5   63                                                    LA                4.5   79                                                    Mean              5.2   71                                                    ______________________________________                                    

It will appear from Table I that oral administration of VD-1827, HClgives rise to similar serum levels of ampicillin as obtained after anequimolar dose of pivampicillin. It also appears from Table I that theurinary recovery of ampicillin after administration of VD-1827, HCl iscomparable to that following administration of pivampicillin.

As indicated in Table II, only 5.2% of penicillanic acid 1,1-dioxidewere excreted in the urine after oral administration of thecorresponding potassium salt. In contrast thereto administration of anequimolar amount of VD-1827, HCl gave a 71% urinary recovery ofpenicillanic acid 1,1-dioxide, thus again illustrating the efficientoral absorption of VD-1827, HCl.

By using the compounds of the invention the antibacterial spectrum ofthe penicillin in question is widely extended, as also β-lactamaseproducing strains will be susceptible to treatment. As mentioned above,such β-lactamase producing strains are found with increasing frequencyand are a serious problem in the clinical therapy. The compounds of theinvention will for such purpose be of extreme value.

Therapeutically the new compounds have distinct advantages over merecombinations of the penicillins and the β-lactamase inhibitors to whichthey are hydrolyzed, or combinations of orally active esters thereof.

For example, many of the β-lactamase inhibitors, including penicillanicacid 1,1-dioxide, are absorbed poorly or irregularly from thegastro-intestinal tract (cf. Table II). Also, many of the penicillins,including ampicillin and carbenicillin, are incompletely absorbed. Inaddition, individual variations in the rate of absorption of the variouspenicillins and β-lactamase inhibitors may in many instances lead to asituation where the active components are not present simultaneously orin the optimum ratio, even if the two drugs are given simultaneously.

Certain easily hydrolyzable esters of penicillins and β-lactamaseinhibitors are absorbed better from the gastro-intestinal tract than thecorresponding free acids. However, hydrolysis of such esters in theorganism gives rise to the formation of inactive by-products, andalthough these by-products are relatively non-toxic, it is undesirableto expose the organism to unnecessary metabolites. Another disadvantageby using combinations of easily hydrolyzable esters of the penicillinsand the β-lactamase inhibitors is that the ester moieties increase themolecular weight of the compounds and consequently the size of thedosage unit. By using the compounds of the invention, the size of thedosage units can be decreased considerably.

In addition, the absorption of such esters will normally not take placesimultaneously, even if the compounds are given to the patient at thesame time. For instance, the pivaloyloxymethyl ester of ampicillin isbeing absorbed very rapidly, whereas the sparingly solublepivaloyloxymethyl ester of the β-lactamase inhibitor penicillanic acid1,1-dioxode is being absorbed much more slowly.

All of these disadvantages are avoided by using the compounds of theinvention.

It has been found that the in vitro synergy between the differentβ-lactamase inhibitors and various penicillins is particularlypronounced when the ratio between the two components is between 3:1 and1:3. As the various penicillins have slightly different biologicalhalf-lives and distribution characteristics, the ratio between theliberated components of the new compounds in the organs and tissues mayvary to some degree, but will normally be within the above preferredlimits.

The invention also comprises methods for the preparation of thecompounds described above.

According to one method of the invention a compound of formula V:##STR3## in which R₁, R₃, and A are as defined above, B stands for anazido group, a protected amino group, e.g. a benzyloxycarbonylamino,triphenylmethylamino, 1-methoxycarbonylpropen-2-yl-amino or1-N,N-dimethylaminocarbonylpropen-2-yl-amino group, or a protectedcarboxy group, such as a benzyloxycarbonyl or cyanomethoxycarbonylgroup, or similar known protected amino or carboxy groups, is subjectedto a hydrogenolysis or hydrolysis depending on what A and B stand for.

The reactions are performed in mixtures consisting of a suitable organicsolvent, e.g. ethyl acetate or tetrahydrofurane, and water, in a ratioof 3:1 to 1:3, preferably 1:1, and at temperatures from 0° to 30° C. IfB is an azido group or another group which can be converted into anamino or carboxy group by hydrogenolysis, e.g. palladium on carbon maybe used as a catalyst, and if B is a group susceptible to hydrolysis,this may be catalyzed by acid, e.g. hydrochloric, hydrobromic orsulphuric acid or p-toluenesulphonic acid.

The intermediates of formula V may be prepared by reacting a compound offormula VI: ##STR4## in which R₁, R₃, and B have the meanings as definedabove, and X stands for a leaving group, such as a halogen atom, with acompound of formula A-M in which A is as defined before and M is acation, such as Na⁺, K⁺, an ammonium ion, a tri- or a tetraalkylammoniumion, e.g. a tetrabutylammonium ion.

The reaction is performed in a suitable solvent, e.g. dimethylformamide,ethyl acetate, dichloromethane, acetone or hexamethyl phosphoric acidtriamide, for a sufficient time and at an adequate temperature with aview to accomplish the desired conversion, usually at a temperature from0° to 60° C.

Another method for the preparation of the intermediates of formula Vcomprises a first step in which a compound A-M is reacted with acompound of formula VII to afford an intermediate of formula VIII;##STR5## in which formulae R₃, and X are as defined above, and Y is abromine or iodine atom, an alkylsulphonyloxy, arylsulphonyloxy,chlorosulphonyloxy, or α-haloalkoxysulphonyloxy radical, Y being abetter leaving group than X.

Surprisingly suitable are compounds of formula VII in which Y is thechlorosulphonyloxy radical, in particular chloromethyl chlorosulphate,because the use hereof gives rise to high yields of chloromethyl estersand avoids the formation of undesired by-products such as A-CH₂ -A.

The reaction is performed in the same manner as described for thepreparation of the known compounds of formula VI and takes place in asuitable solvent, e.g. dimethylforamide, ethyl acetate, dichloromethane,acetone or hexamethylphosphoric acid triamide, usually at a temperaturefrom 0° to 60° C.

When using e.g. chloromethyl chlorosulphate, the process is withadvantage performed under phase transfer conditions, e.g. by using areaction medium consisting of methylene chloride/water containing asuitable neutralizing agent together with a phase transfer catalyst,e.g. (C₄ H₉)₄ N⁺ HSO₄ ⁻.

In a second step the intermediate of formula VIII is reacted with apenicillin derivative of formula IX: ##STR6## in which R₁, B, and M aredefined above to form the intermediate of formula V. If desired, the Xin formula VIII can in advance be exchanged by a better leaving group.

Such a better leaving group is the iodo group. The iodo group isintroduced by using an iodide which is soluble in the reaction mediumused and the cation of which forms a sparingly soluble salt with theleaving group to be exchanged. From a technical point of view, theiodoalkyl esters of formula VIII in which X is an iodo group are keyintermediates in the preparation of the compounds of formula I,resulting in high yields and purity of the end products.

A suitable reaction medium is in particular a lower aliphatic ketone,e.g. acetone or 2-butanone. A preferred iodide is sodium iodide, butother iodides fulfilling the above conditions may also be used.

Another embodiment of the method comprises a first step in which acompound of formula A-M is reacted with a 6-aminopenicillanic acid esterof formula X or an amino-produced derivative thereof, e.g. atrialkylsilyl derivative, to afford a compound of formula IX: ##STR7##in which formulae R₃, A, and X are as defined before. The reaction isperformed in a suitable organic solvent, e.g. dimethylformamide, and attemperatures between 0° and 30° C.

Alternatively, the intermediates of formula XI can be prepared byreacting 6-aminopenicillanic acid or a salt or an amino-protectedderivative thereof with a compound of formula VIII.

In a second step a compound of formula XI or a trialkylsilyl derivativethereof is reacted with a reactive derivative of an acid of formula XII:##STR8## in which R₁ and B are as defined above. B can in addition beNH₃ ⁺, Hal⁻. The reactive derivative can for instance be an acid halide,such as an acid chloride or acid bromide; an acid anhydride; a mixedanhydride with an alkyl-carbonic acid, a carboxylic acid, an inorganicacid or a sulphonic acid; or a radical obtained by reacting the freeacid of formula XII with a carbodiimide or N,N'-carbonyl-diimidazole ora similarly functioning compound. The reaction can be performed in anorganic solvent or in a mixture thereof with water at low or slightlyelevated temperature. Suitable solvents are dichloromethane, chloroform,ethyl acetate, acetone, dimethylformamide, dimethylacetamide, ether,dioxane or other inert solvents.

The starting materials or intermediates of formula V, VIII and XI areunknown compounds and are also within the scope of the presentinvention.

A further embodiment of the method, by which the compounds of formula I,R₂ being a primary amino group, can be prepared directly by a one-stepprocedure, comprises reacting a salt of an aminopenicillin, e.g.ampicillin or amoxycillin, represented by the general formula XIII:##STR9## with a compound of formula VIII, in which formulae R₁, R₃, M, Aand X are as defined before, and preferably X stands for an iodine atom.The reaction is performed in a suitable organic solvent, e.g. ethylacetate, dichloromethane, chloroform, dimethylformamide, and attemperatures between 0° and 40° C., preferably at room temperature.

The starting materials of formulae VI, VII, IX, and X are known or maybe prepared by methods analogous to those used for the preparation ofsimilar known compounds.

Most of the starting materials of formula A-M or the corresponding acidsare known compounds. New compounds are acids and salts corresponding toA being a radical of formula II in which R₅ stands for certain acylaminoradicals. The latter compounds are penicillin sulphones, which may beprepared by known methods.

The compounds of formula I can be purified and isolated in usual mannerand may be obtained either as such or in the form of a salt.

The compounds may in some cases be obtained as diastereomeric mixtureswhich when desired may be separated by known methods, e.g.chromatography.

It is a further object of the present invention to providepharmaceutical compositions which are useful in the treatment ofinfectious diseases in the human and veterinary practice, and which maybe used for enteral, parenteral or topical administration.

With this object in view, the compositions of the invention contain asan active component at least one member selected from the groupconsisting of compounds of the formula I and salts thereof as definedabove, together with solid or liquid pharmaceutical carriers and/ordiluents.

In the said compositions, the proportion of therapeutically activematerial to carrier substance can vary between 1% and 95% by weight. Thecompositions can be worked up to various pharmaceutical forms ofpresentation, such as tablets, pills, dragees, suppositories, capsules,sustained-release tablets, suspensions and the like containing thecompounds of formula I or their antoxic salts, as defined above, mixedwith carriers and/or diluents.

Pharmaceutically acceptable, non-toxic, organic or inorganic, solid orliquid carriers, and/or diluents can be used to make up compositionscontaining the present compounds. Gelatine, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, buffers or other known carriers, auxiliary agents and/ordiluents for medicaments are all suitable. Furthermore, the compositionsmay contain other therapeutically active components which canappropriately be administered together with the present compounds in thetreatment of infectious diseases, such as other antibacterials,antitussiva, pain-relieving drugs, probenecid, etc. In particular,antibacterials, which act synergistically with one or both of the activecomponents formed by in vivo hydrolysis of the compounds of theinvention, are appropriate.

The compounds of formula I can be used either as such or in the form ofa salt. The compounds as such are only slightly soluble in water,whereas many of the salts, e.g. the hydrochlorides and the sodium salts,are readily soluble in water.

Thus the free base VD 1827 is well suited for pharmaceuticalpreparations by being in crystalline form and stable and in additionreadily absorbable as will appear from Table III.

                  TABLE III                                                       ______________________________________                                        Urinary excretion of ampicillin (A) and penicillanic                          acid sulfone (B) in healthy, semi-fasting volunteers                          following oral administration of 170 mg of VD-1827,                           free base (equimolar to 100 mg of anhydrous ampicillin)                       in aqueous suspension.                                                               Urinary Excretion (% of Administered Dose)                                    0-3 HOURS 3-6 HOURS   0-6 HOURS                                        Subject  A      B        A    B      A    B                                   ______________________________________                                        WOG      60.7   67.4     8.3  11.1   69.0 78.5                                VD       50.4   57.8     6.7   9.3   57.1 67.1                                KH       40.0   45.9     12.7 14.1   52.7 60.0                                SV       58.1   65.0     6.9  14.0   65.0 70.0                                MEAN     52.3   59.0     8.7  12.1   61.0 71.1                                ______________________________________                                    

As indicated above, the present compounds may be worked up topharmaceutical forms of presentation including suspensions andnon-aqueous ointments. A pharmaceutical preparation for oral treatmentmay be in the form of a suspension of one of the present compounds, thepreparation containing from 10 mg to 100 mg per ml of the vehicle.

Another object of the invention resides in the selection of a dose ofthe compounds of the invention and a dosage unit of the compositions ofthe invention which dose and dosage unit can be administered so that thedesired activity is achieved without simultaneous secondary effects. Inthe human therapy, the present compounds are conveniently administered(to adults) in dosage units of the compositions containing not less than50 mg and up to 2500 mg, preferably from 100 mg to 1000 mg calculated asthe compound of formula I.

By the term "dosage unit" is meant a unitary, i.e. a single dose whichis capable of being administered to a patient, and which may be readilyhandled and packed, remaining as a physically stable unit dosecomprising either the active material as such or a mixture of it withsolid or liquid pharmaceutical diluents, carriers, solvents and/orauxiliary agents.

In the form of a dosage unit, the compound may be administered once ormore times a day at appropriate intervals, always depending, however, onthe condition of the patient, and in accordance with the prescriptionmade by the medical practitioner.

Thus a daily dose will preferably be an amount of from 0.25 to 15 g of acompound of formula I or an equivalent amount of a salt thereof asdefined before, which conveniently can be divided into several singledoses.

In the continuous therapy of patients suffering from infectiousdiseases, the tablets or capsules are the appropriate form ofpharmaceutical preparation, if desired in the form of sustained-releaseformulations.

In the veterinary practice the above pharmaceutical compositions mayalso be used, preferably in the form of dosage units containing from 50mg up to 25 g of the compound of formula I or a corresponding amount ofa salt thereof.

For the treatment of mammary disorders, especially bovine mastitis, theantibacterial agent can be administered by the intramammary route inliquid or semiliquid form, such as an ointment, or together with asubstantially water-insoluble and oil-insoluble binding agent in theform of granules.

Still another object of the invention is to provide a method of treatingpatients suffering from infectious diseases, the method comprisingadministering to adult patients an effective amount of a compound offormula I, either as such or in the form of a salt as defined before,and preferably, in the form of the dosage units aforesaid. The compoundsof formula I are typically administered in amounts of 3-200 mg/kg bodyweight of the patient/day, corresponding to, for adult human patients,from 0.25 g to 15 g per day, or an equivalent amount of a salt asdefined before of a compound of formula I.

In the treatment of patients, the present compounds can be administeredeither alone or together with other therapeutically active compounds,e.g. probenecid, which aid in combatting the bacterial infection. Suchcombined treatment can be performed with formulations containing more orall of the therapeutically active compounds, or these may beadministered in separate formulations, these being given simultaneouslyor with suitable intervals.

In the treatment of patients, the daily dose is administered either atone time, or in divided dosages, e.g. two, three or four times a day.

In the following "Preparations" the methods for preparing new startingmaterials and intermediates are more specifically described.

Preparation 1 6α-Bromopenicillanic acid 1,1-dioxide

To a stirred solution of potassium permanganate (1.90 g, 12 mmol) inwater (35 ml) and acetic acid (1.36 ml, 24 mmol) was added dropwise at0°-5° C. an icecold solution of potassium 6α-bromopenicillanate (1.91 g,6 mmol) in water (25 ml). After the addition was finished (about 15minutes), the mixture was stirred for another 20 minutes at the lowtemperature. The cooling-bath was removed, and to the mixture was addedsolid sodium pyrosulphite (1.52 g, 8 mmol) to reduce excess oxidationreagent. Precipitated manganese oxides were filtered off, and to thefiltrate (about 60 ml) was added solid sodium chloride (20 g) and ethylacetate (50 ml). The pH of the mixture was adjusted to 1.5 by additionof 4 N hydrochloric acid with stirring, and the organic phase wasseparated. The aqueous phase was reextracted with ethyl acetate (25 ml),and the combined organic extracts were washed with saturated aqueoussodium chloride, dried, and evaporated in vacuo. The amorphous residuethus obtained was crystallized from ether-diisopropyl ether to afford6α-bromopenicillanic acid 1,1-dioxide, melting point: 124°-127° C.

A crystalline potassium salt of the above compound was obtained byaddition of 1 M potassium 2-ethylhexanoate in acetone (3.6 ml) to astirred solution of 6α-bromopenicillanic acid 1,1-dioxide (0.94 g, 3mmol) in acetone (12 ml).

The NMR spectrum of potassium 6α-bromopenicillanate 1,1-dioxide (CD₃ OD)showed signals at δ=1.48 (s, 3H; 2-CH₃), 1.59 (s, 3H; 2-CH₃), 4.48 (s,1H; 3-H), 5.10 (d, J=2Hz, 1H; 6-H), and 5.35 (d, J=2Hz, 1H; 5-H) ppm.Tetramethylsilane was used as internal reference.

PREPARATION 2 6α-Chloropenicillanic acid 1,1 -dioxide

By substituting potassium 6α-chloropenicillanate for the potassium6α-bromopenicillanate in the procedure of Preparation 1,6α-chloropenicillanic acid 1,1-dioxide was obtained as crystals fromdiisopropyl ether, melting point: 134°-137° C.

The NMR spectrum (CDCl₃) showed signals at δ=1.50 (s, 3H; 2-CH₃), 1.64(s, 3H; 2-CH₃), 4.46 (s, 1H; 3-H), 4.70 (d, J=1.5Hz, 1H; 6-H), and 5.18(d, J=1.5Hz, 1H; 5-H) ppm Tetramethylsilane was used as internalreference.

A crystalline potassium salt of the above compound was obtained byaddition of an equimolar amount of 0.8 M potassium 2-ethylhexanoate inacetone to a stirred solution of 6α-chloropenicillanic acid 1,1-dioxidein acetone.

Preparation 3 Chloromethyl penicillanate 1,1-dioxide

To a solution of penicillanic acid 1,1-dioxide (1.17 g, 5 mmol) indimethylformamide (7.5 ml) was added triethylamine (0.98 ml, 7 mmol) andchloroiodomethane (2.18 ml, 30 mmol), and the mixture was stirred atroom temperature for 4 hours. After dilution with ethyl acetate (30 ml),the mixture was washed with water (3×10 ml) followed by saturatedaqueous sodium chloride (5 ml), dried, and evaporated in vacuo to leavethe desired compound as a yellowish oil, which crystallized fromether-petroleum ether, melting point: 94°-96° C.

The NMR spectrum (CDCl₃) showed signals at δ=1.47 (s, 3H; 2-CH₃), 1.66(s, 3H; 2-CH₃), 3.53 (d, J=3Hz, 2H; 6α-H and 6β-H), 4.46 (s, 1H; 3-H),4.68 (t, J=3Hz, 1H; 5-H), and 5.85 (ABq, J=6Hz, 2H; OCH₂ Cl) ppm.Tetramethylsilane was used as internal reference.

Preparation 4 1-Chloroethyl penicillanate 1,1-dioxide

Following the procedure of Preparation 3, but substituting1-chloro-1-iodoethane for the chloroiodomethane and increasing thereaction time to 16 hours, crude 1-chloroethyl penicillanate 1,1-dioxidewas obtained as a yellow oil which could be purified by dry columnchromatography on silica gel (ethyl acetate-petroleum ether, 7:3).

Preparation 5 Chloromethyl 6α-bromopenicillanate 1,1-dioxide

By substituting 6α-bromopenicillanic acid 1,1-dioxide for thepenicillanic acid 1,1-dioxide in the procedure of Preparation 3,chloromethyl 6α-bromopenicillanate 1,1-dioxide was obtained as ayellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.48 (s, 3H; 2-CH₃), 1.64(s, 3H; 2-CH₃), 4.46 (s, 1H; 3-H), 4.71 (d, J=1.5 Hz, 1H; 6-H), 5.17 (d,J=1.5 Hz, 1H; 5-H), and 5.80 (ABq, J=6 Hz, 2H; OCH₂ Cl) ppm. TMS wasused as internal reference.

Preparation 6 Chloromethyl 6β-bromopenicillanate

By substituting potassium 6β-bromopenicillanate for the penicillanicacid 1,1-dioxide and the triethylamine in the procedure of preparation3, chloromethyl 6β-brompenicillanate was obtained as a viscous oil.

Preparation 7 Chloromethyl clavulanate

Following the procedure of Preparation 3, but substituting sodiumclavulanate for the penicillanic acid 1,1-dioxide and the triethylamine,chloromethyl clavulanate was obtained.

Preparation 8 Chloromethyl penicillanate 1,1-dioxide

To a suspension of potassium penicillante 1,1-dioxide (1.08 g) indimethylformamide (12 ml) was added bis-chloromethyl sulphate (1.6 g),and the mixture was stirred at room temperature for 45 minutes. Afterdilution with ethyl acetate (50 ml), the mixture was washed with waterfollowed by aqueous sodium bicarbonate, dried and evaporated in vacuo toleave an oil which was purified by chromatography on silica gel to yieldthe desired compound, identical with the compound described inpreparation 3.

Preparation 9 Chloromethyl 6α-chloropenicillanate 1,1-dioxide

By substituting 6α-chloropenicillanic acid 1,1-dioxide for thepenicillanic acid 1,1-dioxide in the procedure of Preparation 3,chloromethyl 6α-chloropenicillanate 1,1-dioxide was obtained as aviscous oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.48 (s, 3H; 2-CH₃), 1.64(s, 3H; 2-CH₃), 4.47 (s, 1H; 3-H), 4.68 (d, J=1.5 Hz, 1H; 6-H), 5.17 (d,J=1.5 Hz, 1H; 5-H), and 5.81 (ABq, J=6 Hz, 2H; OCH₂ Cl) ppm. TMS wasused as internal reference.

Preparation 10 Iodomethyl penicillanate 1,1-dioxide

To a solution of chloromethyl penicillanate 1,1-dioxide (5.6 g, 20 mmol)in acetone (45 ml) was added sodium iodide (9 g), and the mixture wasstirred at room temperature for 16 hours. Precipitated sodium chloride(1.15 g) was filtered off, the solvent was removed in vacuo, and theresidue thus obtained was treated with ethyl acetate-ether (1:1).Insoluble sodium iodide (6 g ) was filtered off, and the filtrate wasevaporated at reduced pressure.

The residual oil was purified by column chromatography on silica gel(ethyl acetate-n-hexan, 4:6) to yield the title compound as colourlesscrystals from ether, melting point: 101°-102° C.

Preparation 11 6β-Aminopenicillanic acid 1,1-dioxide hydrate A.6β-Benzyloxycarbonylaminopenicillanic acid 1,1-dioxide

To a stirred solution of 6β-benzyloxycarbonylaminopenicillanic acid(63.5 g) and potassium hydrogen carbonate (18.1 g) in water (1125 ml)was slowly (about 45 minutes) at 0° C. added a solution of potassiumpermanganate (38 g) in water (915 ml). During the oxidation, a pH of 6.5was maintained in the reaction mixture by addition of dilute sulphuricacid. Insoluble material was removed by filtration, and the filtrate wasextracted with ethyl ether. The resulting aqueous phase was filteredagain and, after addition of ethyl acetate (600 ml), acidified to pH 2.5with stirring. The organic layer was separated, and the aqueous phasewas extracted with addition ethyl acetate (2×300 ml). After drying, thecombined ethyl acetate extracts were evaporated in vacuo. The residuewas recrystallized from ethyl acetate (250 ml)-petroleum ether (500 ml)to yield the pure compound, melting point: 153°-154° C.; [α]_(D) ²⁰:+146.9°(c=1, 96% C₂ H₅ OH).

B, 6β-Aminopenicillanic acid 1,1-dioxide hydrate

A filtered solution of 6β-benzyloxycarbonylaminopenicillanic acid1,1-dioxide (15.3 g) and potassium hydrogen carbonate (4 g) in water(160 ml) was hydrogenated over 10% Pd/BaSO₄ (5 g) for 4 hours atslightly elevated pressure. After filtration and extraction with ethylether (100 ml), the pH of the ice-cold aqueous solution was adjusted to2.5. The precipitate thus formed was-filtered off, washed with water,and air-dried. Recrystallization from dimethylformamide-water affordedthe pure monohydrate; melting point: 199°-200° C. (dec.); ]α]_(D) ²⁰:+252.9°(c=1, deimethylformamide).

Preparation 12 Chloromethyl 1,1-dioxopenicillanate

To a mixture of potassium 1,1-dioxopenicillanate (2.7 g, 10 mmol),potassium hydrogen carbonate (6.0 g, 60 mmol) and tetrabutylammoniumhydrogen sulphate (0.34 g, 1 mmol) in water (10 ml) and dichloromethane(15 ml), chloromethyl chlorosulphate (1.5 ml) was added. After stirringfor 1 hour at 30° C., the mixture was filtered and the organic layer wasseparated and dried (sodium sulphate). After dilution with propanol-2(25 ml), the solution was concentrated to about 10 ml in vacuo and leftat 5° C. for 1 hours. The crystals were filtered off, washed with coldpropanol-2 and dried in vacuo to give the title compound as colourlesscrystals with a melting point of 94°-96° C.

Preparation 13 1-Chloroethyl 1,1-dioxopenicillanate

To a mixture of potassium 1,1-dioxopenicillanate (40.7 g, 0.15 mol),silver nitrate (25.5 g, 0.15 mol), and silver oxide ( 7.5 g) inacetonitrile ( 750 ml), 1-chloro-1-iodoethane (42 ml) was added. Afterstirring for 48 hours at ambient temperature, the silver salts werefiltered off, and the filtrate taken to dryness in vacuo. The residuewas dissolved in ethyl acetate (200 ml), and the solution was washedwith saturated aqueous, sodium chloride, filtered, dried, and evaporatedin vacuo. Chromatography of the residue on silica gel (hexane-ethylacetate, 3:2) gave the title compound as a crystalline mixture of thetwo diastereomers with m.p. 130°-132° C.

Preparation 14 1-Iodoethyl 1,1-dioxopenicillanate

To a solution of 1-chloroethyl 1,1-dioxopenicillanate (30 g,˜0.1 mol) inacetone (100 ml), sodium iodide (30 g, 0.2 mol) was added, and themixture was stirred at ambient temperature for 3 days. Aqueous sodiumthiosulphate was added, and the acetone was removed in vacuo. Theseparated oil was dissolved in ethyl acetate, and the solution waswashed with water, dried and evaporated in vacuo. The residual oil waschromatographed on silica gel (hexane-ethyl acetate, 3:1) to give acrystalline mixture (m.p. 134°-36° C.) of the diastereomeric 1-iodoethyland 1-chloroethyl esters, containing 40% of the iodo compound, accordingto the microanalytical determination of iodine.

Preparation 15 Chloromethyl 6β-bromopenicillanate

To a stirred solution of potassium 6β-bromopenicillanate (0.96 g, 3mmol) and potassium bicarbonate (1.80 g, 18 mmol) in water (9 ml) andethyl acetate (9 ml) was added tetrabutylammonium hydrogen sulphate(0.10 g, 0.3 mmol), followed by chloromethyl chlorosulphonate (0.45 ml,4.5 mmol), and the mixture was stirred at room temperature for 1.5hours. The organic phase was separated, and the aqueous phasereextracted with ethyl acetate (9 ml). The combined organic extractswere washed with water (2×5 ml), dried, and concentrated to about 5 mlat reduced pressure. The concentrate was subjected to dry columnchromatography on silica gel (petroleum ether-ethyl acetate, 9:1) toafford pure chloromethyl 6β-bromopenicillanate as an almost colourlessoil.

The NMR spectrum (CDCl₃) showed signals at δ=1.54 (s, 3H; 2-Ch₃), 1.70(s, 3H; 2-CH₃), 4.54 (s, 1H; 3-H), 5.35 and 5.59 (2d, J=4Hz, 2H; 5-H and6-H), and 5.77 (ABq, J=5Hz, 2H; OCH₂ Cl) ppm. Tetramethylsilane was usedas internal reference.

Preparation 16 Iodomethyl 6β-bromopenicillanate

To a solution of chloromethyl 6β-bromopenicillanate (0.82 g, 2.5 mmol)in acetone (5 ml) was added solid sodium iodide (0.75 g, 5.0 mmol), and,after protection from light, the mixture was stirred at room temperaturefor 24 hours. Precipitated sodium chloride was filtered off, washed withacetone (2×1 ml), and the filtrate was evaporated in vacuo to leave anoily residue which was redissolved in ethyl acetate (20 ml). Theresulting solution was washed with water (2×10 ml), dried (MgSO₄), and,following concentration to about 5 ml at reduced pressure, subjected tocolumn chromatography on silica gel using petroleum ether-ethyl acetate,9:1, as the eluent. Fractions containing the pure title compound, asrevealed by thinlayer chromatography (TLC), were combined and evaporatedin vacuo to yield iodomethyl 6β-bromopenicillanate as a slightlyyellowish oil.

The NMR spectrum showed signals at δ=1.55 (s, 3H; 2-CH₃), 1.69 (s, 3H,2-CH₃), 4.50 (s, 1H; 3-H), 5.34 and 5.57 (2d, J32 4Hz, 2H; 5-H and 6H),and 5.97 (ABq, J=5Hz, 2H; OCH₂ I) ppm. Tetramethylsilane was used asinternal reference.

Preparation 17 Chloromethyl1,1-dioxo-6β-(2,6-dimethoxybenzamido)penicillanate

Chloromethyl chlorosulphate (1.8 ml, 18 mmol) was added during 20minutes at room temperature to a mixture of1,1-dioxo-6β-(2,6-dimethoxybenzamido)penicillanic acid (methicillinsulpone; 6.2 g, 15 mmol), potassium hydrogen carbonate (8.7 g, 87 mmol)and tetrabutylammonium hydrogen sulphate (0.51 g, 1.5 mmol) in water (15ml) and dichloromethane (15 ml).

After stirring for a further 15 minutes, the organic phase wasseparated, dried, and evaporated in vacuo to leave an oil whichcrystallized from 96% ethanol to yield colourless crystals with m.p.142°-143° C. (dec). Two recrystallizations from acetone-water gave theanalytical sample with m.p. 154°-155° C. (dec); [α]_(D) ²⁰ :+195°(c=1,CHCl₃).

Preparation 18 Iodomethyl1,1dioxo-6β-(2,6-dimethoxybenzamido)penicillanate

Sodium iodide (3 g, 20 mmol) was added to a solution of chloromethyl1,1-dioxo-6β-(2,6-dimethoxybenzamido)penicillanate (2.31 g, 5 mmol) inacetone (10 ml), and the mixture was stirred overnight at roomtemperature. Addition of water precipitated the title compound ascrystals which were collected by filtration and dried in vacuo; m.p.153°14 156° C. (dec).

The product was dissolved in a mixture of acetone and 96% ethanol, theacetone was removed in vacuo and the desired compound crystallized. Byrepeating this procedure the m.p. was raised to 169°-170° C. (dec.);[α]_(D) ²⁰ :+197°)c=1, CHCl₃).

Preparation 19 Chloromethyl 1,1-dioxo-6β-chloropenicillanate

By substituting potassium 1,1-dioxo-6α-chloropenicillanate for thepotassium 6β-bromopenicillanate in the procedure of Preparation 15, thetitle compound was obtained as colourless crystals fromether-diisopropyl ether; melting point: 111°-113° C; [α]_(D) ²⁰+210°(c=0.5, CHCl₃).

Preparation 20 Iodomethyl 1,1-dioxo-6αchloropenicillanate

By substituting chloromethyl 1,1dioxo-6αchloropenicillanate for thechloromethyl 6β-bromopenicillanate in the prodedure of Preparation 16,the title compound was obtained as a colourless foam.

The NMR spectrum (CDCl₃) showed dignals as δ=1.49 (s, 3H; 2-CH₃), 1.62(s, 3H; 2-CH₃), 4.41 (s, 1H; 3-H), 4.66 and 5.16 (2d, J=1.5 Hz, 2H; 5-Hand 6-H), and 6.01 (ABq, J=5 Hz, 2H; OCH₂ I) ppm. Tetramethylsilane wasused as internal reference.

Preparation 21 Chloromethyl 1,1-dioxo-6α-bromopenicillanate

By substituting potassium 1,1-dioxo-6α-bromopenicillanate for thepotassium 6β-bromopenicillanate in the producure of Preparation 15, thetitle compound was obtained as colourless crystals fromether-diisopropyl ether; melting point: 92°-93° C; [α]_(D) ²⁰+185°(c=0.5, CHCl₃).

Preparation 22 Iodomethyl 1,1-dioxo-6αbromopenicillanate

By substituting chloromethyl 1,1-dioxo-6α-bromopenicillanate for thechloromethyl 6β-bromopenicillanate in the procedure of Preparation 16,the title compound was obtained as a colourless foam which failed tocrystallize.

The NMR spectrum (CDCl₃) showed signals at δ=1.49 (s, 3H; 2-CH₃), 1.63(s, 3H; 2-CH₃), 4.41 (s, 1H; 3-H), 4.70 and 5.16 (2d, J=1.5 Hz, 2H; 5-Hand 6-H), and 6.01 (ABq, J=5 Hz, 2H; OCH₂ I) ppm. Tetramethylsilane wasused as internal reference.

Preparation 23 Chloromethyl 6β-iodopenicillanate

By substituting potassium 6β-iodopenicillanate for the potassium6β-bromopenicillanate in the procedure of Preparation 15, the titlecompound was obtained as a slightly yellowish oil.

The NMR spectrum (CDCl₃) showed signals as δ=1.52 (s, 3H; 2-CH₃), 1.71(s, 3H; 2-CH₃), 4.55 (s, 1H; 3-H), 5.40 and 5.63 (2d, J=3.5 HZ, 2H; 5-Hand 6-H), and 5.78 ABq, J=5.5 Hz, 2H; OCH₂ Cl) ppm. Tetramethylsilanewas used as internal reference.

Preparation 24 Iodomethyl 6β-iodopenicillanate

By substituting chloromethyl 6β-iodopenicillanate for the chloromethyl6β-bromopenicillanate in the procedure of Preparation 16, the titlecompound was obtained as a yellowish oil.

The NMR spectrum (CDCl₃) showed singles at δ=1.53 (s, 3H; 2-CH₃), 1.70(s, 3H; 2-CH₃), 4.53 (s, 1H; 3-H), 5.39 and 5.61 (2d, J=3.5 Hz, 2H; 5-Hand 6-H), and 6.00 (ABq, J=5.5 Hz, 2H; OCH₂ I) ppm. Tetramethylsilanewas used as internal reference.

Preparation 25 Chloromethyl 6β-chloropenicillanate

By substituting potassium 6β-chloropenicillanate for the potassium6β-bromopenicillanate in the procedure of Preparation 15, the titlecompound was obtained as a colourless oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.53 (s, 3H; 2-CH₃), 1.69(s, 3H; 2-CH₃), 4.54 (s, 1H; 3-H), 5.24 and 5.62 (2d, J=4 Hz, 2H; 5-Hand 6-H), and 5.80 (ABq, J=5 Hz, 2H; OCH₂ Cl) ppm. Tetramethylsilane wasused as internal reference.

Preparation 26 Iodomethyl 6β-chloropenicillanate

By substituting chloromethyl 6β-chloropenicillanate for the chloromethyl6β-bromopenicillanate in the procedure of Preparation 16, the titlecompound was obtained as a slightly yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.52 (s, 3H; 2-CH₃), 1.69(s, 3H; 2-CH₃), 4.52 (s, 1H; 3-H), 5.22 and 5.58 (2d, J=4 Hz, 2H; 5-Hand 6-H), and 5.99 (ABq, J=5 Hz, 2H; OCH₂ I) ppm. Tetramethylsilane wasused as internal reference.

Preparation 27 Chloromethyl 6β-bromopenicillanate A. Chloromethyl6,6-dibromopenicillanate

By substituting potassium 6,6-dibromopenicillanate for the potassium6β-bromopenicillanate in the procedure of Preparation 15, the titlecompound was obtained as a slightly yellowish oil which crystallizedfrom ether-diisopropyl ether; melting point: 105°-107° C.; [α]_(D) ²⁰:+206° (c=0.5, CHCl₃).

The NMR spectrum (CDCl₃) showed signals at δ=1.54 (s, 3H; 2-CH₃), 1.66(s, 3H; 2-CH₃), 4.60 (s, 1H; 3-H), 5.80 (ABq, J=5 Hz, 2H; OCH₂ Cl), and5.83 (s, 1H; 5-H) ppm. Tetramethylsilane was used as internal reference.

B. Chloromethyl 6β-bromopenicillanate

To a stirred solution of chloromethyl 6,6-dibromopenicillanate (1.63 g,4 mmol) in dry benzene (40 ml) was added under nitrogen at 0° C.tri-n-butyltin hydride (1.16 g, 4 mmol). After stirring at roomtemperature for 18 hours, the mixture was evaporated in vacuo. Theresidual oil was purified by dry column chromatography on silica gel(petroleum ether-ethyl acetate, 85:15) to yield pure chloromethyl6β-bromopenicillanate as a slightly yellowish oil.

The NMR spectrum of the product was identical with that of the compounddescribed in Preparation 15.

Preparation 28 Bromomethyl 1,1-dioxopenicillanate

To a solution of sodium bromide (1.0 g) in N,N-dimethylformamide (10 ml)was added chloromethyl 1,1-dioxopenicillanate (0.28 g, 1 mmol), and themixture was stirred at room temperature for 20 hours. After dilutionwith ethyl acetate (50 ml), the mixture was washed with water (4×10 ml),dried, and evaporated in vacuo. The residue was purified by columnchromatography on silica gel to yield the desired compound as ayellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.49 (s, 3H; 2-CH₃), 1.64(s, 3H; 2-CH₃), 3.52 (m, 2H; 6-H, 4.47 (s, 1H; 3-H), 4.75 (m, 1H; 5-H),and 5.98 (ABq, J=4.5 HZ, 2H; OCH₂ Br) ppm. TMS was used as internalreference.

Preparation 29 VD 1827, tosylate

VD 1827, hydrochloride (6.31 g, 10 mmole) was dissolved in a mixture ofwater (40 ml) and acetone (10 ml). A solution of sodium4-toluenesulfonate (1.94 g, 10 mmole in water (10 ml) was added dropwisewith stirring. After stirring for 1 hour at room temperature and 2 hoursat 5° C., the crystalline precipitate was collected, washed with water(2×10 ml) and dried in vacuo to yield the title compound as colourlesscrystals with melting point 141°-148° C. dec.

Water content (K.F. method): 5.1%.

The IR-spectrum (KBr) showed strong bands at: 1790, 1680, 1515, and 1325cm⁻¹.

The NMR-spectrum [(CD₃)₂ SO] showed signals at δ=1.37 (s, 6H), 1.48 (s,6H), 2.32 (s, 3H), 3.1-3.8 (m, 2H), 4.47 (s, 1H), 4.57 (s, 1H), 5.2 (m,2H), 5.6 (m, 2H), 5.95 (s, 2H), 7.15 (d, J=7.5, 2H), 7.5 (m, 7H), 8.7(bs, 3H), 9.45 (d, J=6, 1H) ppm. Tetramethylsilane was used as internalreference.

Preparation 30 Preparation of VD 1827, tosylate

To a suspension of potassium carbonate (1.66 g, 12 mmole) indimethylformamide (25 ml), were added methyl acetoacetate (2.38 ml, 22mmole) and anhydrous ampicillin (3.84 g, 11 mmole). The mixture wasstirred for 3 hours at room temperature, followed by 18 hours at 5° C.Iodomethyl penicillanate 1,1-dioxide (3.73 g, 10 mmole) was added, andstirring was continued for 20 minutes at 5°-10° C. After dilution withethyl acetate (100 ml), the mixture was extracted with water (4×25 ml)and saturated aqueous sodium chloride (25 ml) to give a solution of theintermediate,6β-[N-(1-methoxycarbonyl-propen-2-yl)-D-α-amino-α-phenylacetamido]penicillanoyloxymethylpenicillanate 1,1-dioxide, in ethyl acetate. The intermediate washydrolyzed at an apparent pH-value of 1 (glass-calomel combinationelectrode) by dropwise addition of a solution of 4-toluenesulfonic acidmonohydrate (1.90 g, 10 mmole) in ethyl acetate (20 ml). After additionof about 5 ml of the solution, the mixture was seeded, and a crystallineprecipitate was formed. The mixture was stirred for 2 hours at 5° C.,whereafter the crystals were filtered off, washed with ethyl acetate(2×10 ml), and air-dried.

The dried product was dissolved in methanol (40 ml) at 40° C. Water (80ml) was added, and crystallization was induced by scratching and coolingin an ice-bath. After stirring for 1 hour at 5° C., the crystals werefiltered off, washed with water (2×10 ml), and dried in vacuo to givethe title compound as colourless crystals, melting point: 141°-148° C.

The invention will be further described in the following Examples whichare not to be construed as limiting the invention.

EXAMPLE 1 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride A.1,1-Dioxopenicillanoyloxymethyl6-(D-α-azido-α-phenylacetamido)penicillanate

To a solution of chloromethyl6-(D-α-azido-α-phenylacetamido)penicillanate (2.54 g, 6 mmol) indimethylformamide (35 ml) was added potassium penicillanate 1,1-dioxide(1.63 g, 6 mmol), and the mixture was stirred at room temperature for 20hours. After dilution with ethyl acetate (140 ml), the mixture waswashed with water (4×35 ml), followed by saturated aqueous sodiumchloride (20 ml), and the organic phase was dried and evaporated invacuo. The yellow oily residue thus obtained was purified by dry columnchromatography on silica gel (cyclohexane-ethyl acetate, 1:1) to yieldthe desired compound as a yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.43 (s, 3H; 2-CH₃), 1.52(s, 3H; 2-CH₃), 1.59 (s, 3H; 2-CH₃), 1.66 (s, 3H; 2-CH₃), 3.48 (d, J=3Hz, 2H; 6α-H and 6β-H), 4.44 (s, 1H; 3-H), 4.51 (s, 1H; 3-H), 4.63 (t,J=3 Hz, 1H; 5-H), 5.13 (s, 1H; CHN₃), 5.65 (m, 2H; 5-H and 6-H), 5.92(s, 2H; OCH₂ O), and 7.48 (s, 5H; arom. CH) ppm. Tetramethylsilane wasused as internal reference.

B. 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

A solution of 1,1-dioxopenicillanoyloxymethyl6-(D-α-azido-α-phenylacetamido)penicillanate (1.77 g, 2.85 mmol) inethyl acetate (25 ml) was placed in a three-necked flask, equipped witha gas inlet/outlet tube, a glass-calomel combination electrode, and aburette controlled by an automatic titrator. Water (20 ml) and 10%palladium on carbon catalyst (1.77 g) were added, and the system wasflushed with nitrogen. Thereafter, a stream of hydrogen was bubbledthrough the suspension with stirring, a pH-value of 2.5 being maintainedin the aqueous phase by the addition of 0.5 N aqueous hydrochloric acidvia the automatic titrator. When the consumption of acid stopped, theflask was flushed with nitrogen until all hydrogen was removed, and thecatalyst was filtered off. The aqueous phase was separated andfreeze-dried to give the desired compound as a colourless foam.

The NMR spectrum (D₂ O) showed signals at δ=1.38 (s, 6H; 2-CH₃), 1.46(s, 3H; 2-CH₃), 1.58 (s, 3H; 2-CH₃), 3.56 (m, 2H; 6α-H and 6β-H), 4.60(s, 1H; 3-H), 4.63 (s, 1H; 3-H), 5.03 (m, 1H; 5-H), 5.27 (s, 1H;CH-NH₂), 5.53 (s, 2H; 5-H and 6-H), 5.97 (bs, 1H; OCH₂ O), and 7.53 (s,5H; arom. CH) ppm. Tetramethylsilane was used as external reference.

EXAMPLE 2 1,1-Dioxopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate, hydrochlorideA. 1,1-Dioxopenicillanoyloxymethyl6-[N-(benzyloxycarbonyl)-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate

To a solution of chloromethyl penicllanate 1,1-dioxide (1.41 g, 5mmol)^(x)) in dimethylformamide (25 ml) was added potassium6-[N-(benzyloxycarbonyl)-D-α-amino-α-(p-hydroxyphenyl)-acetamido]penicillanate(2.46 g, 5 mmol), and the mixture was stirred at room temperature for 18hours. After dilution with ethyl acetate (100 ml), the mixture waswashed with water (4×25 ml), dried, and evaporated in vacuo. Theresidual oil was purified by dry column chromatography on silica gel(ethyl acetate-petroleum ether 8:2) to yield the desired compound as ayellowish oil.

B. 1,1-Dioxopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate hydrochloride

The benzyloxycarbonyl protecting group of the compound prepared inExample 2A was removed by hydrogenation at atmospheric pressure usingthe method described in Example 1B to afford the title compound as acolourless, amorphous product.

EXAMPLE 3 1-(1,1-Dioxopenicillanoyloxy)ethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

By substituting α-chloroethyl6-(D-α-azido-α-phenylacetamido)penicillanate for the correspondingchloromethyl ester in the procedure of Example 1 A,1-(1,1-dioxopenicillanoyloxy)ethyl6-(D-α-azido-α-phenylacetamido)penicillanate was obtained.

B.

Following the procedure of Example 1 B, but substituting1-(1,1-dioxopenicillanoyloxy)ethyl6-(D-α-azido-α-phenylacetamido)penicillanate for the1,1-dioxopenicillanoyloxymethyl6-(D-α-azido-α-phenylacetamido)penicillanate,1-(1,1-dioxopenicillanoyloxy)ethyl6-(D-α-amino-α-phenylacetamido)penicillanate, hydrochloride was obtainedas an amorphous product.

EXAMPLE 4 1,1-Dioxopenicillanoyloxymethyl6-(D,L-α-carboxy-α-phenylacetamido)penicillanate sodium salt A.1,1-Dioxopenicillanoyloxymethyl6-(D,L-α-benzyloxycarbonyl-α-phenylacetamido)penicillanate

Following the procedure described in Example 2 A, but substitutingsodium 6-(D,L-α-benzyloxycarbonyl-α-phenylacetamido)penicillanate forthe potassium6-[N-benzyloxycarbonyl-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate,the desired compound was obtained.

B. 1,1-Dioxopenicillanoyloxymethyl6-(D,L-α-carboxy-α-phenylacetamido)penicillanate sodium salt

To a solution of 1,1-dioxopenicillanoyloxymethyl6-(D,L-α-benzyloxycarbonyl-α-phenylacetamido)penicillanate (1.43 g, 2mmol) in ethanol (20 ml) was added 10% palladium on carbon catalyst, andthe mixture was hydrogenated at atmospheric pressure until theconsumption of hydrogen ceased. The catalyst was removed by filtration,washed with ethanol, and the filtrate was evaporated in vacuo. The oilresidue thus obtained was dissolved in ethyl acetate (15 ml), water (15ml) was added, and the apparent pH in the aqueous phase was adjusted to7.0 by addition of 0.2 N aqueous sodium hydroxide with stirring. Theaqueous phase was separated and freeze-dried to yield the desiredcompound as a yellowish foam.

EXAMPLE 5 Clavulanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillante hydrochloride

By following the procedure described in Example 1A, but substitutingsodium clavulanate for the potassium penicillanate 1,1-dioxide,clavulanoyloxymethyl 6-(D-α-azido-α-phenylacetamido)penicillanate wasobtained as a yellowish oil.

By catalytic hydrogenation of the above intermediate according to themethod described in Example 1B, the title compound was obtained as anamorphous powder.

EXAMPLE 6 1,1-Dioxo-6α-chloropenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillante hydrochloride

By following the method described in Example 2A, but substitutingchloromethyl 6α-chloropenicillinate 1,1-dioxide for the chloromethylpenicillanate 1,1-dioxide and triethylammonium6-[N-(1-N,N-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanatefor the potassium6-[N-(benzyloxycarbonyl)-D-α-amino-α-(p-hydroxyphenyl)-acetamido]penicillanate,1,1-dioxo-6α-chloropenicillanoyloxymethyl6-[N-(1-N,N-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanatewas obtained.

The protecting group in the above intermediate was removed byacid-catalyzed hydrolysis (pH˜3) in a 1:1 mixture of ethyl acetate andwater to afford, after separation and freeze-drying of the resultingaqueous phase, the title compound as an amorphous product.

EXAMPLE 7 6β-Bromopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

By following the method described in Example 2A, but substitutingchloromethyl 6β-bromopenicillanate for the chloromethyl penicillanate1,1-dioxide and triethylammonium6-[N-(1-N,N-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanatefor the potassium6-[N-(benzyloxycarbonyl)-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate,6β-bromo-penicillanoyloxymethyl6-[N-(1-N,N-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanatewas obtained.

The protecting group in the above intermediate was removed byacid-catalyzed hydrolysis (pH˜3) in a 1:1 mixture of ethyl acetate andwater to afford, after separation and freeze-drying of the resultingaqueous phase, the title compound as an amorphous product.

EXAMPLE 8 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride A.Tetrabutylammonium 6-(D-α-amino-α-phenylacetamido)penicillanate

To a stirred, cooled (5° C.) mixture of6-(D-α-amino-α-phenylacetamido)penicillanic acid trihydrate (8.08 g) andtetrabutylammonium hydrogen sulphate (6.9 g) in water (20 ml) anddichloromethane (40 ml) was added slowly 2 N aqeuous sodium hydroxide(20 ml). The organic layer was separated, and the aqueous phase wasextracted with dichloromethane (20 ml). The combined dichloromethanelayers were dried (MgSO₄) and evaporated in vacuo to leave a viscousoil. The oil was dissolved in ethyl acetate (100 ml), and residualdichloromethane was removed at reduced pressure. After standingovernight at 5° C., the precipitated crystals were collected, washedwith ethyl acetate, and dried in vacuo to give the title compound ascolourless, slightly hygroscopic crystals with melting point 125°-130°C. (decomp.).

1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

To a stirred suspension of tetrabutylammonium6-(D-α-amino-α-phenylacetamido)penicillanate (2.95 g) in ethyl acetate(20 ml) and dichloromethane (5 ml) was added a solution of iodomethyl1,1-dioxopenicillanate (1.9 g) in ethyl acetate (10 ml). After a fewminutes, an almost clear solution was obtained. Dichloromethane wasremoved at reduced pressure and precipitated tetrabutylammonium iodidewas filtered off. From the filtrate the title compound was transferredto an aqueous phase (25 ml) with 1 N aqueous hydrochloric acid (pH 3.0,5° C.), and from the aqueous phase back to an organic phase (ethylacetate, 25 ml) with 0.5 M aqueous sodium hydrogen carbonate (pH 7.0, 5°C.). The organic layer was washed with water and the desired compoundwas again transferred to an aqueous phase as described above. To theaqueous phase was added n-butanol, and the water was removedazeotropically by distillation in vacuo to give the title compound ascolourless crystals, melting point 175°-177° C. (decomp.); [α]_(D) ²⁰:+201° (c=1, H₂ O)

EXAMPLE 9 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate

To a cold (5° C.) solution of the compound prepared in Example 8 (631mg) in water (10 ml), ethyl acetate (10 ml) was added, and the pH of themixture was adjusted to 7.0 by addition of 0.5 M aqueous sodium hydrogencarbonate with stirring. The organic layer was separated, washed withwater, dried (MgSO₄), and evaporated in vacuo to give the title compoundas a colourless solid.

The IR spectrum (KBr) showed strong bands at 1780 and 1690 cm⁻¹.

EXAMPLE 10 1,1-Dioxopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillante hydrochloride A.Tetrabutylammonium6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate

To stirred, cooled (5° C.) solution of tetrabutylammonium hydrogensulphate (3.57 g, 10.5 mmol) in water (10 ml), a mixture ofdichloromethane and n-butanol (9:1, 20 ml) was added, followed by 2Nsodium hydroxide to bring the pH to about 3. Amoxycillin.sup.(x)trihydrate (4.2 g, 10 mmol) was added, and the pH adjusted to 9 with 2Nsodium hydroxide. The organic layer was separated, and the aqueous phasewas extracted twice with 10 ml portions of dichloromethane:n-butanol(9:1). The combined extracts were concentrated to a viscous oil invacuo, and the residue was dissolved in ethyl acetate (50 ml).Crystallization was induced by scratching, and, after standing at 5° C.for 2 hours, the crystals were filtered off, washed and dried to givethe title compound with a melting point of 148°-151° C. (decomp.).

1,1-Dioxopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate, hydrochloride

To a stirred cooled (5° C.) solution of tetrabutylammonium

6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate (606 mg, 1 mmol)in acetonitrile (5 ml), iodomethyl 1,1-dioxopenicillanate (373 mg, 1mmol) dissolved in acetonitrile (2 ml) was added. After stirring for 10minutes at 50° C. ethyl acetate (50) was added, and the solvent wasstripped in vacuo. The residue was dissolved in ethyl acetate (20 ml),and crystallized tetrabutylammonium iodide was removed by filtration. Tothe filtrate, water (10 ml) was added, and the pH was adjusted to 3 withN hydrochloric acid: The aqueous phase was separated and freeze-dried togive the title compound as a colourless powder.

The NMR spectrum [(CD₃)₂ SO] showed signals at δ=1.37 (s, 6H; 2-CH₃),1.50 (s, 6H; 2-CH₃), 3.46 (m, 2H, 6α-H and 6β-H), 4.46 (s, 1H; 3-H),4.57 (s, 1H; 3-H), 5.04 (bs, 1H; CHNH₂), 5.27 (m, 1H; 5-H), 5.58 (m, 2H;5-H and 6-H), 5.96 (bs, 2H, OCH₂ O), 6.87 and 7.37 (2d, J=8.5Hz, 4H;arom. CH) ppm. TMS was used as internal reference.

EXAMPLE 11 1-(1,1-Dioxopenicillanoyloxy)ethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

To a solution of tetrabutylammonium6-(D-α-amino-α-phenylacetamido)penicillanate (5.9 g, 10 mmol) indichloromethane (10 ml) and ethyl acetate (40 ml), 1-iodoethyl1,1-dioxopenicillanate (10.55 g of 40% purity, corresponding to 4.22 g,10.9 mmol) dissolved in ethyl acetate (30 ml) was added. The clearsolution was immediately seeded with tetrabutylammonium iodide,whereafter dichloromethane was removed in vacuo, and separatedtetrabutylammonium iodide was filtered off. From the filtrate, the titlecompound was transferred to an aqueous phase (50 ml) with N hydrochloricacid (pH 3.0, 5° C.) and from the aqueous phase to an organic phase(ethyl acetate, 50 ml) with sodium hydrogen carbonate (pH 7.0, 5° C.).The organic phase was washed with water, and the title compound wasagain transferred to an aqueous phase as described above. Freeze-dryingof the aqueous phase gave the title compound as a colourless powder.

The NMR spectrum (D₂ O) showed signals at δ=1.38 s, 6H; 2-CH₃), 1.43 (s,3H; 2-CH₃), 1.55 (s, 3H; 2-CH₃), 1.56 (d, 3H; CHCH₃), 3.50 (m, 2H; 6α-Hand 6β-H), 4.53 (s, 1H; 3-H), 4.55 and 4.59 (2s, 1H; 3-H), 4.96 (m, 1H;5-H), 5.26 (s, 1H; CHNH₂), 5.51 (s, 2H; 5-H and 6-H), 6.95 (m, 1H;CHCH₃), and 7.51 (s, 5H; arom. CH) ppm.

EXAMPLE 12 6β-Bromopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillante hydrochloride

To a stirred solution of tetrabutylammonium6-(D-α-amino-α-phenylacetamido)penicillanate (0.82 g; 1.4 mmol) in amixture of ethyl acetate (2.8 ml) and dichloromethane (1.4 ml) was addeda solution of iodomethyl 6β-bromopenicillanate (0.60 g, 1.4 mmol) inethyl acetate (5.6 ml). After stirring at room temperature for a fewminutes, crystalline tetrabutylammonium iodide began to precipitate. Thedichloromethane was removed from the rection mixture at reducedpressure, and the crystals were filtered off and washed with ethylacetate (2×2.5 ml). The filtrate was washed with water (5 ml), to theorganic phase was added fresh water (10 ml), and the pH of the aqueousphase was adjusted to 3.1 by addition of 1 N hydrochloric acid withstirring. The aqueous phase was separated and freeze-dried to give thedesired compound as a colourless foam.

The NMR spectrum (D₂ O) showed signals at δ=1.34 (s, 3H; 2-CH₃), 1.36(s, 3H; 2-CH₃), 1.43 (s, 3H; 2-CH₃), 1.58 (s, 3H; 2-CH₃), 4.54 (s, 1H;3-H), 4.75 (s, 1H; 3-H), 5.24 (s, 1H; CHNH₂), 5.46-5.62 (m, 4H; 5-H and6-H), 5.88 (bs, 2H; OCH₂ O), and 7,47 (s, 5H; arom. CH) ppm.

EXAMPLE 13 1,1-Dioxo-6α-chloropenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

By substituting iodomethyl 1,1-dioxo-6α-chloropenicillanate for theiodomethyl 6β-bromopenicillanate in the procedure of Example 12 , thetitle compound was obtained as a colourless foam.

The NMR spectrum (D₂ O) showed signals at δ=1.35 (s, 6H, 2-CH₃), 1.41(s, 3H; 2-CH₃); 1.53 (s, 3H; 2-CH₃), 4.57 (s, 1H; 3-H), 4.73 (s, 1H;3-H), 5.08 (s, 1H; 5-H or 6-H); 5.26 (s, 1H; CHNH₂), 5.34 (s, 1H; 5-H or6-H), 5.49 (s, 2H; 5-H and 6-H), 5.94 (b, 2H; OCH₂ O), and 7.49 (s, 5H;arom. CH) ppm.

EXAMPLE 14 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride A.1,1-Dioxo-6α-bromopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

By substituting iodomethyl 1,1-dioxo-6α-bromopenicillanate for theiodomethyl 6β-bromopenicillanate in the procedure of Example 12, thedesired compound was obtained as a colourless foam.

The NMR spectrum (D₂ O) showed signals at δ=1.36 (s, 6H; 2-CH₃), 1.41(s, 3H, 2-CH₃), 1.54 (s, 3H; 2-CH₃), 4.57 (s, 1H; 3-H), 4.71 (s, 1H,3-H), 5.09 (s, 1H; 5-H or 6-H), 5.27 (s, 1H, CHNH₂), 5.35 (s, 1H; 5-H or6-H), 5.50 (s, 2H; 5-H and 6-H), 5.9 (b, 2H; OCH₂ O), and 7.50 (s, 5H;arom. CH) ppm.

B. 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

To a solution of 1,1-dioxo-6α-bromopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate (liberated from 1.36 g ofthe corresponding hydrochloride) in ethyl acetate (50 ml) was addedwater (25 ml) and 10% palladium on carbon catalyst (0.7g), and themixture was shaken in a hydrogen atmosphere for 40 minutes. Afterremoval of the catalyst by filtration, the pH of the aqueous phase wasadjusted to 2.5 with 1 N hydrochloric acid. From the separated aqueousphase the title compound was transferred to an organic phase (ethylacetate, 25 ml) with aqueous potassium bicarbonate (pH 7.0, 5° C.) andback to a fresh aqueous phase with 1 N hydrochloric acid (pH 2.7). Theaqueous phase was freeze-dried to give the title compound as acolourless powder.

The NMR spectrum of the product was identical with that of the compounddescribed in Example 1.

EXAMPLE 15 6β-Iodopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

Following the procedure described in Example 12 but substitutingiodomethyl 6β-iodopenicillanate for the iodomethyl6β-bromopenicillanate, the title compound was obtained as a colourlessfoam.

The NMR spectrum (D₂ O) showed signals at 67 =1.33 (s, 3H; 2-CH₃), 1.38(s, 3H; 2-CH₃), 1.45 (s, 3H; 2-CH₃), 1.60 (s, 3H, 2-CH₃), 4.56 (s, 1H,3-H), 4.74 (s, 1H; 3-H), 5.22 (s, 1H; CHNH₂), 5.3-5.7 (m, 4H; 5-H and6-H), 5.92 (bs, 2H; OCH₂ O), and 7.49 (s, 5H; arom. CH) ppm.

EXAMPLE 16 6β-Chloropenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

Following the procedure described in Example 12, but substitutingiodomethyl 6β-chloropenicillanate for the iodomethyl6β-bromopenicillanate, the title compound was obtained as a colourlessfoam.

The IR spectrum (KBr) showed strong bands at 1790-1770 and 1690 cm⁻¹.

EXAMPLE 17 Clavulanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride A. Iodomethyl6-(D-α-azido-α-phenylacetamido)penicillanate

To a solution of chloromethyl6-(D-α-azido-α-phenylacetamido)penicillanate (1.32 g, 3 mmol) in acetone(25 ml), sodium iodide (1.80 g, 12 mmol) was added, and the mixture wasstirred at room temperature for 18 hours. The precipitate was filteredoff, and the filtrate was evaporated in vacuo. The residue was extractedwith ethyl acetate (25 ml), the extract was concentrated to about 3 mland subjected to column chromatography on silica gel using hexane ethylacetate 1:1 as eluent. Fractions containing the desired compound werecombined and evaporated in vacuo to leave the title compound as ayellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.58 (s, 3H; 2-CH₃), 1.67(s, 3H; 2-CH₃), 4.47 (s, 1H; 3-H), 5.13 (s, 1H; CHN₃), 5.52-5.82 (m, 2H;5-H and 6-H), 6.00 (ABq, 2H; OCH₂ J), 7.4 (s, 5H, arom. CH), and 7.0-7.4(m, 1H; CONH) ppm. TMS was used as internal reference.

B. Clavulanoyloxymethyl 6-(D-α-azido-α-phenylacetamido)penicillanate

To a solution of iodomethyl 6-(D-α-azido-α-phenylacetamido)penicillanate(378 mg, 0.73 mmol) in hexamethyl phosphoric acid triamide (3.8 ml),lithium clavulanate (90 mg, 0.44 mmol) was added, and the mixture wasstirred at room temperature for one hour. The mixture was diluted withethyl acetate (90 ml) and washed with water (3×20 ml) followed bysaturated aqueous sodium chloride (10 ml), dried, and evaporated invacuo. The yellow oil thus obtained was purified by columnchromatography on silica gel using hexane-ethyl acetate 1:4 as eluent toyield the desired compound as a slightly yellowish foam.

The NMR spectrum (CDCl₃) showed signals at δ=1.51 (s, 3H; 2-CH₃), 1.64(s, 3H; 2-CH₃), 3.11 (d, J=17 Hz, 1H; 6-H), 3.51 (dd, J₁ =17 Hz, J₂ =3Hz, 1H; 6-H), 4.25 (d, J=7 Hz, 2H; CH₂ OH); 4.51 (s, 1H; 3-H), 4.92 (m,1H; ═CH--), 5.13 (s, 1H; 5-H), 5.13 (s, 1H; 3-H), 5.5-5.8 (m, 3H; 5-H,6-H, and CHN₃), 5.89 (ABq, 2H; OCH₂ O), 7.16 (d, J=8.5 Hz, 1H; CONH),and 7.41 (m, 5H; arom. CH) ppm. TMS was used as internal standard.

C. Clavulanoyloxymethyl 6-(D-α-amino-α-phenylacetamido)penicillanatehydrochloride.

A solution of clavulanoyloxymethyl6-(D-α-azido-α-phenylacetamido)penicillanate (130 mg, 0.22 mmol) inethyl acetate (20 ml) was placed in a three-necked flask, equipped witha gas inlet/outlet tube, a glass-calomel combination electrode, and aburette. Water (20 ml) and 10% palladium/on/carbon catalyst (130 mg)were added, and the system was flushed with nitrogen. Hydrogen waspassed through the stirred mixture, and the pH-value was maintained at2.5 by simultaneous addition of 0.1 N aqueous hydrochloric acid. Whenthe consumption of acid ceased, the flask was flushed with nitrogen, andthe catalyst was filtered off. The aqueous layer was separated, filteredand freeze-dried to give the desired compound as a colourless powder.

The NMR spectrum [(CD₃)₂ SO] showed signals at δ=1.30 (s, 3H; 2-CH₃),1.44 (s, 3H; 2-CH₃), 3,12 (d, J=17 Hz, 1H; 6-H), 3.65 (dd, J₁ =17 Hz, J₂=3 Hz, 1H; 6-H), 4.00 (m, 2H; CH₂ OH), 4.42 (s, 1H; 3-H), 4.75 (m, 1H;--CH═), 5.15 (bs, 1H; 3-H), 5.40-5.75 (m, 3H; 5-H, 6-H, and CHNH₂) ,5.85 (ABq, 2H; OCH₂ O), 7.50 (m, 5H; arom. CH), and 9.45 (d, J=7 Hz, 1H;CONH) ppm. TMS was used as internal reference.

EXAMPLE 18 Clavulanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate hydrochloride A.Chloromethyl6-[N-benzyloxycarbonyl-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate

To a suspension of potassium6-[N-benzylocycarbonyl-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate(2.46 g, 5 mmol in N,N-dimethylformamide (25 ml) was addedchloroiodomethane (2.18 ml), 30 mmol), and the mixture was stirred atroom temperature for 3 hours. After dilution with ethyl acetate (100ml), the mixture was washed with water (4×25 ml), dried, and evaporatedin vacuo. The residue was purified by column chromatography on silicagel (using ethyl acetate/hexane 1:1 as eluent) to yield the desiredcompound as a yellowish oil.

B. Clavulanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate hydrochloride

By following the procedure described in Example 17 A, 17 B, and 17 C butsubstituting chloromethyl6-[N-benzyloxycarbonyl-D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanatefor the chloromethyl 6-(D-α-azido-α-phenylacetamido)penicillanate, thetitle compound was obtained as a colourless freeze-dried powder.

The IR spectrum (KBr) showed strong bands at 1775 and 1690 cm⁻¹.

EXAMPLE 19 1,1-Dioxopenicillanoyloxymethyl6-(D,L-α-carboxy-α-phenylacetamido)penicillanate sodium salt A. 1,1Dioxopenicillanoyloxymethyl6-(D,L-α-benzyl-oxycarbonyl-α-phenylacetamido)penicillanate

To a suspension of sodium6-(D,L-α-benzyloxycarbonyl-α-phenylacetamido)penicillanate (0.98 g, 2mmol) in N,N-dimethylformamide (10 ml), iodomethyl penicillanate1,1-dioxide (0.75 g, 2 mmol) was added, and the mixture was stirred for30 min. at room temperature. Ethyl acetate (50 ml) was added, and themixture was extracted with saturated aqueous calcium chloride (3×12 ml),dried, and evaporated in vacuo. The oily residue was purified by columnchromatography on silica gel using hexane-ethyl acetate 1:1 as eluent toyield the desired compound as a yellowish oil.

The NMR spectrum (CDCl₃) showed signals at δ=1.4-1.6 (m, 12H; 2-CH₃),3.46 (m, 2H; 6-H), 4.4-4.5 (m, 2H; 3-H and CHCO), 4.56-4.65 (m, 2H; 3-Hand 5-H), 5.19 (s, 2H; PhCH₂ O), 5.4-5.75 (m, 2H; 5-H and 6-H), 5.9(ABq, 2H; OCH₂ O), 7.3 (s, 5H; arom. CH), 7.35 (s, 5H; arom. CH), and7.5-7.95 (m, 1H; CONH) ppm. TMS was used as internal reference.

B. 1,1-Dioxopenicillanoyloxymethyl6-(D,L-α-carboxy-α-phenylacetamido)penicillanate sodium salt

To a solution of 1,1-dioxopenicillanoyloxymethyl6-(D,L-α-benzyloxycarbonyl-α-phenylacetamido)penicillanate (1.0 g, 1.4mmol) in ethyl acetate (25 ml), water (25 ml) and 10% palladium oncarbon catalyst (1.0 g) were added, and the pH-value of the mixture wasadjusted to 7.0. Hydrogen was bubbled through the stirred mixture, andthe pH-value was maintained at 7.0 by addition of 0.1 N aqueous sodiumhydroxide. When the consumption of base stopped (after about 1 hour),the catalyst was filtered off, and the aqueous phase was separated,filtered, and freeze-dried to give the desired compound as a colourlesspowder.

The NMR-spectrum (D₂ O) showed signals at δ=1.47 (s, 3H; 2-CH₃), 1.53(s, 3H; 2-CH₃), 1.63 (s, 6H; 2-CH₃), 3.55 (m, 2H; 6-H), 4,12 (s, 1H;3-H), 4,17 (s, 1H; 3-H), 4.70 (s, 1H; CHCO), 5.00 (m, 1H; 5-H), 5.4-5.7(m, 2H; 5-H and 6-H), 6.00 (bs, 2H; OCH₂ O), 7.42 (s, 5H; arom. CH) ppm.TMS was used as external reference.

EXAMPLE 20 1,1-Dioxopenicillanoyloxymethyl 6β-aminopenicillanatehydrochloride A. Tetrabutylammonium 6β-aminopenicillanate

To a stirred, ice-cooled mixture of 6β-aminopenicillanic acid (4.32 g,20 mmol), tetrabutylammonium hydrogen sulphate (6.8 g, 20 mmol),dichloromethane (50 ml), and water (20 ml) was added slowly a solutionof sodium hydroxide (1.60 g, 40 mmol) in water (3.5 ml). The organiclayer was separated, and the aqueous layer was extracted withdichloromethane (2×25 ml). The combined organic layers were dried andevaporated in vacuo to leave the desired compound as a viscous oil.

The IR spectrum (CHCl₃) showed strong bands at 1760 and 1610 cm⁻¹.

B. 1,1-Dioxopenicillanoyloxymethyl 6β-aminopenicillanate hydrochloride

To a solution of tetrabutylammonium 6β-aminopenicillanate (5.1 g. 11mmol) in ethyl acetate (25 ml) was added a solution of iodomethylpenicillanate 1,1-dioxide (3.73 g, 10 mmol) in ethyl acetate (25 ml).After stirring for 15 min. at room temperature, the precipitate wasfiltered off, and the filtrate was evaporated in vacuo. The residue waspurified by column chromatography on Sephadex ®LH 20 usingchloroform-hexane 65:35 as eluent. The purified product was dissolved inethyl acetate (25 ml), water (25 ml) was added, and the pH-value of themixture was adjusted to 2.0 by addition of 2 N hydrochloric acid.

The aqueous phase was separated and freeze-dried to give the titlecompound as a colourless powder.

The NMR spectrum (D₂ O) showed signals at δ=1.52 (s, 3H; 2-CH₃), 1.60(s, 3H; 2-CH₃), 1.65 (s, 3H; 2-CH₃), 1.76 (s, 3H; 2-CH₃), 3.52-3.8 (s,2H; 6-H), 4.78 (s, 1H; 3-H), 4.90 (s, 1H; 3-H), 5.05-5.25 (m, 1H; 5-H),5.20 (d, J=4 Hz, 1H; 6-H), 5.78 (d, J=4 Hz, 1H; 5-H), and 6.08 (bs, 2H;OCH₂ O) ppm. TMS was used as external reference.

Example 21 1,1-Dioxopenicillanoyloxymethyl6-(d-α-amino-α-phenylacetamido)penicillanate hydrochloride

To a stirred suspension of D-α-phenylglycyl chloride hydrochloride (1.98g, 10 mmol) in dichloromethane (25 ml) was added at 0° C. sodiumhydrogen carbonate (1.68 g, 20 mmol) followed by1,1-dioxopenicillanoyloxymethyl 6-aminopenicillanate hydrochloride (3.98g, 8 mmol). After vigorous stirring at 0° C. for 1.5 h, the mixture wasevaporated in vacuo. The residue was taken up in an ice-cooled mixtureof ethyl acetate (25 ml) and saturated aqueous sodium hydrogen carbonate(25 ml). The organic phase was separated, water (20 ml) was added, andthe pH-value of the mixture was adjusted to 2.5 by addition of 2Nhydrochloric acid. The aqueous phase was separated and freeze-dried togive an amorphous powder which crystallized from ethanol-butanone-2 toyield a product identical with that described in Example 8.

Example 22 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride A. Potassium6-[N-(1-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanate

To a solution of triethylammonium6-[N-(1-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanate(27.3 g, 48 mmol) in acetone (1 liter), 1 M potassium 2-ethylhexanoatein acetone (49 ml) was added dropwise. After stirring at roomtemperature for 2 hours, the precipitate was filtered off andrecrystallized from methanol-isopropanol to afford the title compound;melting point: 201-203° C. (dec.); [α]_(D) ²⁰ =+174° (c=1, water).

B. 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride

To an ice-cooled solution of potassium6-[N-(1-dimethylaminocarbonylpropen-2-yl)-D-α-amino-α-phenylacetamido]penicillanate(5.49 g, 11 mmol) in N,N-dimethylformamide (25 ml), iodomethylpenicillanate 1,1-dioxide (3.73 g, 10 mmol) was added, and the mixturewas stirred at 5° C. for 30 minutes. After dilution with ethyl acetate(100 ml), the mixture was extracted with water (4×25 ml) and saturatedaqueous sodium chloride (25 ml). The organic phase was dried andevaporated in vacuo to half the initial volume. Water (25 ml) was added,and the apparent pH-value of the mixture was adjusted to 2.5 by additionof 2N hydrochloric acid with stirring. During the hydrolysis thispH-value was maintained by addition of further hydrochloric acid. Whenthe consumption of acid ceased (after about 30 minutes), the aqueousphase was separated and freeze-dried to give a compound, which aftercrystallization from ethanol/butanone-2 was identical with thatdescribed in Example 8.

EXAMPLE 23 1,1-Dioxo-6β-(2,6-dimethoxybenzamido)penicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate hydrochloride.

Sodium 6-(D-α-amino-α-phenylacetamido)penicillanate (0.75 g, 2 mmol) wasadded to an ice-cold solution of iodomethyl1,1-dioxo-6β-(2,6-dimethoxybenzamido)penicillanate (1.11 g, 2 mmol) indimethylformamide (10 ml). The resulting solution was kept in anice-bath for 30 minutes, diluted with ethyl acetate (40 ml) and washedwith water (4×10 ml). The organic phase was stirred with water whilehydrochloric acid was added to pH 2.5. The aqueous phase was separatedand freeze-dried to yield the title compound as a colourless powder.

The NMR spectrum (CD₃ OD, TMS as internal reference) showed signals atδ=1.47 (s, 3H; 2-CH₃), 1.50 (s, 6H; 2-CH₃), 1.58 (s, 3H, 2-CH₃), 3.83(s, 6H; OCH₃), 4.50 (s, 1H; 3-H), 4.69 (s, 1H; 3-H), 5.18 (s, 1H;CHNH₂), 5.21 (d, J=4 Hz, 1H; 5-H), 5.4-5.8 (m, 2H; 5-H and 6-H), 6.00(m, 2H; OCH₂ O), 6.27 (d, J=4 Hz, 1H; 6-H), 6.73 (d, 2H; arom. 3-H and5-H), 7.43 (t, 1H; arom. 4-H), and 7.53 (s, 5H, arom. CH) ppm.

EXAMPLE 24 1-(1,1-Dioxopenicillanoyloxy)ethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido] penicillanate hydrochloride

By following the procedure of Example 10B, but substituting 1-iodoethyl1,1-dioxopenicillanate for the iodomethyl 1,1-dioxopenicillanate, thetitle compound was obtained as a colourless powder.

The IR spectrum (KBr) showed strong bands at 1785, 1690, and 1655 cm⁻¹.

EXAMPLE 25 6β-Bromopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate hydrochloride

By substituting iodomethyl 6β-bromopenicillanate for the iodomethyl1,1-dioxopenicillanate in the procedure of Example 10B, the titlecompound was obtained as a slightly yellowish powder.

The IR spectrum (KBr) showed strong bands at 1790, 1775, and 1690 cm⁻¹.

EXAMPLE 26 6β-Iodopenicillanoyloxymethyl6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanate hydrochloride

By following the procedure described in Example 10B, but substitutingiodomethyl 6β-iodopenicillanate for the iodomethyl1,1-dioxopenicillanate, the title compound was obtained as an amorphouspowder.

The IR-spectrum (KBr) showed strong bands at 1790, 1775, and 1685 cm⁻¹.

EXAMPLE 27 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate

VD-1827 Tosylate (38.4 g, 0.05 mole) was dissolved in acetonitrile (250ml), and the solution was cooled in an icebath. Diisopropylethylamine(9.1 ml, 0.05 mole) and isopropanol (500 ml) were added, and the mixturewas seeded to initiate crystallization. After stirring for 20 minutes,further isopropanol (500 ml) was added dropwise during 1 hour. Stirringwas continued for 2 hours at 5° C., whereafter the precipitate wasfiltered off, washed with isopropanol, and dried in vacuo to yield thetitle compound as colourless crystals.

The IR spectrum (KBr) showed strong bands at: 3400, 3300, 1800, 1785,1755, 1680 and 1510 cm⁻¹.

The NMR-spectrum (CD₃ CN) showed signals at δ-1.40 (s, 3H); 1.50 (s,3H); 1.53 (s, 3H); 1.63 (s, 3H); 2.08 (bs, 2H); 3.25 (dd, J=16.6 Hz andJ=2.0 Hz, 1H); 3.57 (dd, J=16 Hz and J=4.4 Hz, 1H); 4.43 (s, 1H); 4.48(s, 1H); 4.52 (s, 1H); 4.77 (dd, J=2.0 Hz and J=4.4 Hz, 1H); 5.55 (m,2H); 5.86 (s, 2H); 7.33 (s, 5H); 8.16 (m, 1H) ppm. Tetramethylsilane wasused as internal reference.

EXAMPLE 28 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate

VD-1827 Tosylate (19.17 g, 25 mmole) was suspended in an ice-cooledmixture of water (125 ml) and ethyl acetate (500 ml). Saturated aqueoussodium bicarbonate (50 ml) was added, and the mixture was stirred for 30minutes at 5° C. The organic phase was isolated, dried (MgSO₄), andevaporated to a volume of about 200 ml. Seeding crystals were added, andthe mixture was stirred for 2 hours at 5° C. The crystalline precipitatewas filtered off, washed with ice-cooled ethyl acetate, and dried invacuo to give the title compound.

EXAMPLE 29 1,1-Dioxopenicillanoyloxymethyl6-(D-α-amino-α-phenylacetamido)penicillanate

A suspension of VD 1827, tosylate (26 g) in isopropanol (500 ml) wasstirred for 15 minutes at ambient temperature, Diisopropylethylamine (7ml) was added in three equal portions with intervals of 5 minutes. Themixture was warmed to 33°-35° C. and stirred at this temperature untilthe reaction was complete (about 2 hours). The end point was determinedby microscopical examination of the crystals and was further ensured bya solubility test: a sample was filtered off and washed with isopropanoland ether. A 50 mg sample of the dried product should give a clearsolution when treated with 1 ml 0.1N hydrochloric acid.

The crystals were filtered off and washed successively with isopropanoland ether. The product was dried in the air to give the pure titlecompound as colourless crystals exhibiting an ill-defined melting point(slow decomposition above 150° C.). [α]_(D) ²⁰ +199° (c=1, dioxane).

Found: C, 50.35; H, 5.17; N 9.39; S, 10.78. C₂₅ H₃₀ N₄ O₉ S₂ requires C,50.49; H, 5.09; N, 9.42 and S, 10.78%.

What we claim is:
 1. A method for producing a compound of formula I##STR10## in which R₁ stands for a phenyl, 4-hydroxyphenyl,1,4-cyclohexadienyl or a 3-thienyl group; R₂ represents a primary aminoor a carboxy group; R₃ is a hydrogen atom, or a lower alkyl, aryl oraralkyl radical, and A stands for a radical of a β-lactamase inhibitorcontaining a β-lactam ring as well as a carboxy group, A being connectedvia the carboxy group, and salts of the compounds of formula I withpharmaceutically acceptable, non-toxic acids or bases, said Asubstituent being a radical selected from the group consisting of##STR11## in which R₄ stands for a hydrogen or a halogen atom; R₅ is ahydrogen atom or an amino or acylamino group, but at least one of R₄ andR₅ is hydrogen; R₆ represents a halogen atom; and R₇ stands for ahydroxyl group, or one of the radicals of known clavulanic acidderivatives with β-lactamase inhibitory activity, wherein a compound offormula V: ##STR12## in which R₁, R₃, and A are as defined above, Bstands for an azido group, a protected amino group, or a protectedcarboxy group, is subjected to a catalytic hydrogenolysis or hydrolysisdepending on what A and B stand for.
 2. The method of claim 1, in whichB stands for a member selected from the group consisting ofbenzyloxycarbonylamino, triphenylmethylamino,1-methoxycarbonyl-propen-2-yl-amino,1-N,N-dimethylaminocarbonylpropen-2-yl-amino, benzyloxycarbonyl, andcyanomethoxycarbonyl.
 3. A method for producing a compound of formulaVIII ##STR13## wherein R₃ is hydrogen, A is a radical of a β-lactamaseinhibitor containing a β-lactam ring as well as a carboxy group, A beingconnected via the carboxy group and being a radical selected from thegroup consisting of ##STR14## in which R₄ stands for a hydrogen or ahalogen atom; R₅ is a hydrogen atom or an amino or acylamino group, butat least one of R₄ and R₅ is hydrogen; R₆ represents a halogen atom; andR₇ stands for a hydroxyl group, or one of the radicals of knownclavulanic acid derivatives with β-lactamase inhibitory activity and Xis chlorine comprising reacting chloromethyl chlorosulphate with acompound A-M wherein A is as defined above and M is a cation.
 4. Themethod of claim 3 wherein A is a penicillanic acid 1,1-dioxide radical.5. A method for producing a compound of the formula ##STR15## in whichR₃ is a hydrogen atom, or a lower alkyl, aryl or aralkyl radical, and Astands for a radical of a β-lactamase inhibitor containing a β-lactamring as well as a carboxy group, A being connected via the carboxygroup, and salts of the compounds of formula I with pharmaceuticallyacceptable, non-toxic acids or bases, said A substituent being a radicalselected from the group consisting of ##STR16## in which R₄ stands for ahydrogen or a halogen atom; R₅ is a hydrogen atom or an amino oracylamino group, but at least one of R₄ and R₅ is hydrogen; R₆represents a halogen atom; and R₇ stands for a hydroxyl group, or one ofthe radicals of known clavulanic acid derivatives with β-lactamaseinhibitory activity, and in which X is iodine, comprising reacting thecorresponding chloroalkyl ester with an iodide in a lower aliphaticketone.
 6. The method of claim 5 wherein the iodide is sodium iodide andthe aliphatic ketone is acetone or 2-butanone.
 7. A method for producingiodomethyl penicillanate 1,1-dioxide comprising reacting thecorresponding chloromethyl ester with sodium iodide in acetone.